For image acquisition, approximately 15 worms were transferred onto a 35 mm MatTek glass bottom culture dish (MatTek, Ashland, MA, USA) containing a 6 μL drop of 50 mM sodium azide (NaN3). Confocal images were collected using a Leica TCS SP8 microscope. GFP fluorescence was illuminated using a 488 nm argon laser line and red fluorophors with a 561 nm solid state laser with either a 20x 0.6NA Apochromat air objective, or a 40x 1.3NA oil Apochromat CS2 objective. Images were captured using a spectral NyD detector. DIC images were collected using a transmitted light detector and the 488 nm argon laser line. Confocal images were acquired using LAS AF software (Leica Microsystems) and visualized, rendered, and analyzed using Volocity Software (Perkin Elmer). For quantification of aggregates, images were captured with the 40x objective, from the vulva to tail region of each animal, and analyzed using the Volocity Software. Approximately 10 worms were imaged for each line and the data represented as a scatter plot. Experiments were repeated three times and representative data shown in the figures. To quantify eggs in utero, 40x DIC images of the uterus were collected. Eggs number was counted manually. Experiments were conducted at least 3 times and data were collected from at least 10 animals per line.
Glass bottom culture dish
Manufactured by MatTek
Sourced in United States
The glass bottom culture dish is a laboratory equipment designed to facilitate cell culture and microscopic observation. It consists of a standard cell culture vessel with a transparent glass bottom, allowing for direct visualization of cells under a microscope.
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Lab products found in correlation
Deltavision core microscope system, by Cytiva (5 mentions)
Subcellular protein fractionation kit, by Thermo Fisher Scientific (4 mentions)
Lsm 900 confocal microscope, by Zeiss (3 mentions)
710 confocal microscope, by Zeiss (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Tcs sp8 microscope, by Leica camera (2 mentions)
Volocity software, by PerkinElmer (2 mentions)
C1 confocal microscope, by Nikon (2 mentions)
Eclipse ti, by Nikon (2 mentions)
Softworx software, by Cytiva (2 mentions)
Coolsnap hq2 camera, by Cytiva (2 mentions)
Fm4 64, by Thermo Fisher Scientific (2 mentions)
Fluorescently labeled secondary antibody, by LI COR (2 mentions)
Lysis buffer, by Thermo Fisher Scientific (2 mentions)
Live dead viability cytotoxicity kit, by Thermo Fisher Scientific (2 mentions)
Mouse monoclonal antibody directed against pd l1, by Cell Signaling Technology (2 mentions)
Mouse monoclonal antibody directed against β actin, by Cell Signaling Technology (2 mentions)
Ifn γ, by Thermo Fisher Scientific (2 mentions)
Il 1β, by Thermo Fisher Scientific (2 mentions)
Mvx10 microscope, by Olympus (2 mentions)
42 protocols using glass bottom culture dish
1
Imaging and Quantification of C. elegans
2
Visualizing LGG-1 Puncta in Worms
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For microscopic image acquisition, approximately 12 worms were transferred to a 35 mm MatTek glass bottom culture dish (MatTek, Ashland, MA) containing 6 µl of 50 mM sodium azide. Confocal images were collected using a Leica TCS SP8 microscope and visualized, rendered and analyzed using Volocity Software (v6.11, Perkin Elmer). LGG-1 puncta were quantified using the Threshold Object Identification method in Volocity.
3
Live-cell Imaging of hiPSC
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Lifeact- or PODXL-GFP expressing 1196a hiPSC (see cloning in the Constructs and cell lines section) were plated on six-well plates (Nunc) in mTeSR1/2Si medium, and time-lapse images were taken at 37°C using the IncuCyte Zoom live cell imaging (Sartorius). Alternatively, cells were plated on a glass bottom culture dish (MatTek), and were imaged in a live-cell imaging chamber (Tokai HIT) configured for Olympus IX-83 at 37°C.
4
Visualizing Megakaryocyte Maturation and Platelet Release
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BM explants were obtained by flushing femurs, cutting into 1 mm pieces, and staining with anti–GPIX-AF488 antibody (10 μg/mL) for 30 minutes. Explants were then placed in a glass-bottom culture dish (MatTek) precoated overnight with fibronectin (20 μg/mL; MilliporeSigma) with Tyrode’s buffer (137 mM NaCl; 2 mM KCl; 0.3 mM NaH2PO4; 5.5 mM glucose; 5 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid; 12 mM NaHCO3; 2 mM CaCl2, pH 7.4) and maintained at 37°C in a VivaView FL Incubator Microscope (Olympus). Images were acquired every 5 minutes in differential interference contrast and GFP channels for 18 hours to visualize MKs released at the periphery of the explants. Videos were analyzed using ImageJ (NIH).
5
Visualizing Cav-1 and hnRNPa2b1 Dynamics
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Cells were seeded on the coverglass or glass-bottom culture dish (MatTek), followed by double transfection with cav-1-mCherry and hnRNPa2b1-EGFP. Localization and trafficking of the fluorescence proteins were analyzed using Leica SP5 Confocal Microscope and Zeiss LSM 710-Live Duo Confocal with 2-Photon Capability, respectively.
6
Visualizing Retinal Ganglion Cell Stratification
7
Visualizing Sporulation Dynamics in Yeast
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Cells were induced to sporulate using the resuspension method in Sterlini-Mandelstam (SM) medium (60 (link)). At different time points, 150 µL of cell cultures was harvested and resuspended in 10 µL PBS containing 5 µg mL−1 FM4-64 membrane dye and/or 2 µg mL−1 DAPI to visualize DNA, as needed. Finally, 3 µL of cell suspension was placed on a glass bottom culture dish (MatTek) and covered with 1% agar pad made with SM medium. Cells were viewed using a DeltaVision core microscope system equipped with an environmental chamber at 22 °C. Cell images were captured with a Photometrics cool snap HQ2 camera. Eight planes were acquired every 0.2 µm and the data were submitted to deconvolution using SoftWorx software (61 (link)).
8
Cell Culture Protocols for Breast Cancer Lines
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Cell lines were obtained from ATCC (Manassas, VA, USA) and cultured following ATCC requirement. Cells were seeded on a glass bottom culture dish (35 mm diameter, poly-D-lysine coated; MatTek Corporation) and grown overnight at 37°C in a 5% CO2 incubator. MCF10A cells were cultured in DMEM (Thermo Fisher Scientific, catalog 11320) with 5% horse serum (Thermo Fisher Scientific, catalog 16050), 20 ng/mL EGF, 0.5 mg/mL hydrocortisone, 100 ng/mL Cholera toxin, 10 μg/mL bovine insulin with penicillin/ streptomycin. T47D and MDA231 cells were cultured in DMEM (Thermo Fisher Scientific, catalog 11965-092) with 10% fetal bovine serum (FBS) (ATCC, catalog 30-2020), 4 mM L-glutamine and 10 mM HEPES, pH 7.4.
9
Indirect Co-culture of Cardiomyocytes and Macrophages
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A schematic of the indirect co‐culture model is shown in Figure 1 A. MES‐CM (250,000 cells) were seeded on the glass center of the glass bottom culture dish (MatTek). Macrophages were cultured in a 6‐well plate (40,000 cells/well) and polarized as described above. Conditioned medium from macrophage cultures was then centrifuged (3 min, 10,000 RPM) to remove any debris or cells before mixing in 1:1 ratio with cardiomyocyte differentiation media to culture mES‐CM for up to 72 h.
10
Collagen Matrices for Cell Migration
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Collagen matrices were formed as described previously (Rommerswinkel et al., 2014 ). In brief, 50 µl of 10× MEM (Gibco) and 27 µl of 7.5% sodium bicarbonate (Thermo Fisher) were added to 375 µl of 3.3 mg/ml rat tail type-I collagen (Advanced BioMatrix). From this mixture, 115 µl was added to 50 µl of DMEM-10% FBS containing 104 cells. The resulting collagen concentration is 1.9 mg/ml. Next, 150 µl of the combined collagen-cell mixture was loaded onto the glass portion of a glass-bottom culture dish (Mattek) and allowed to gel at either 21°C or 37°C for LR or HR matrices, respectively. For 2D migration studies, 50 µl collagen-cell mixture was loaded onto the glass portion of the dish to enable feasible working distance for microscopy. For 21°C gelling, dishes were inverted for the first 10–15 min to avoid cell sedimentation before placing right-side up until complete gelling occurred. Dishes were gently flooded with culture medium after 30 min for 37°C gels and 1 h for 21°C gels, and left to equilibrate for 2–3 h. Cells were imaged every 10 min with a 20× objective under 0.5× magnification on an Olympus VivaView FL microscope. Nuclei were stained with the Vybrant DyeCycle Green nuclear stain at the end of experiments.
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